The cholinergic system is involved in regulation of the development of the hematopoietic system.

Gene expression profiling demonstrated that components of the cholinergic system, including choline acetyltransferase, acetylcholinesterase and nicotinic acetylcholine receptors (nAChRs), are expressed in embryonic stem cells and differentiating embryoid bodies (EBs). Triggering of nAChRs expressed in EBs by nicotine resulted in activation of MAPK and shifts of spontaneous differentiation toward hemangioblast. In vivo, non-neural nAChRs are detected early during development in fetal sites of hematopoiesis. Similarly, in vivo exposure of the developing embryo to nicotine resulted in higher numbers of hematopoietic progenitors in fetal liver. However postpartum, the number of hematopoietic stem/progenitor cells (HSPC) was decreased, suggesting an impaired colonization of the fetal bone marrow with HSPCs. This correlated with increased number of circulating HSPC and decreased expression of CXCR4 that mediates migration of circulating cells into the bone marrow regulatory niche. In addition, protein microarrays demonstrated that nicotine changed the profile of cytokines produced in the niche. While the levels of IL1alpha, IL1beta, IL2, IL9 and IL10 were not changed, the production of hematopoiesis-supportive cytokines including G-CSF, GM-CSF, IL3, IL6 and IGFBP-3 was decreased. This correlated with the decreased repopulating ability of HSPC in vivo and diminished hematopoietic activity in bone marrow cultures treated with nicotine. Interestingly, nicotine stimulated the production of IL4 and IL5, implying a possible role of the cholinergic system in pathogenesis of allergic diseases. Our data provide evidence that the nicotine-induced imbalance of the cholinergic system during gestation interferes with normal development and provides the basis for negative health outcomes postpartum in active and passive smokers.

The role of nicotinic acetylcholine receptors in lymphocyte development.

The sizes of lymphocyte populations in lymphoid organs of nicotinic acetylcholine receptor knockout and chimera (knockout/wild-type) mice were studied by flow cytometry. The absence of beta2 subunit decreased, while nicotine treatment increased B lymphocyte numbers in the bone marrow. In chimera mice, either beta2 or alpha7 subunits influenced lymphocyte populations in primary lymphoid organs, while in the spleen, only alpha7 receptors were critical. More annexin V-positive B cells were found in the bone marrow of knockout than wild-type animals. We conclude that nicotinic receptors are involved in regulating lymphocyte development and control the B lymphocyte survival.

Nicotinic receptors regulate B lymphocyte activation and immune response.

The presence of nicotinic acetylcholine receptors (nicotinic receptors) composed of either alpha7 or alpha4 and beta2 subunits is revealed in B lymphocytes by means of radioligand binding assay and Cell ELISA. Mouse B lymphocytes contained 12,200+/-3200 of epibatidine-binding sites and 3130+/-750 of alpha-Bungarotoxin-binding sites per cell. Mice lacking nicotinic receptor subunits alpha4, beta2 or alpha7 had less serum IgG and IgG-producing cells in the spleen, but showed stronger immune response to both protein antigen in vivo and CD40-specific antibody in vitro than wild-type mice. Anti-CD40-stimulated proliferation of B lymphocytes from beta2 knockout, but not wild-type mice was inhibited with nicotine. Our results indicate that signalling through nicotinic receptors affects both the pre-immune state and activation of B lymphocytes in the immune response, possibly via CD40-dependent pathway. This could contribute to immune depression found in tobacco smokers.

The effect of simulated microgravity on hybridoma cells.

The effect of clinostat-simulated microgravity on SP-2/0 and 1D6 hybridoma cells was studied. Clinorotation during 4-5 days at 1.5 rounds per minute decreased dramatically their proliferating capacity: the rotated cells divided less than once while control cells performed 4-5 divisions. They decreased the non-specific adhesion to tissue culture plastic, but increased the number of cell-to-cell contacts. Such phenomenological changes were accompanied with the alterations in pericellular glycosaminoglycans: decreased accumulation of hyaluronic acid and increased accumulation of chondroitin/dermatan-sulfate, as well as with the increase of cytoplasmic Ca2+ concentration. Clinorotation resulted in hybridoma nicotinic receptor desensitization but not down-regulation. In contrast, both the quantity and quality (molecular isoforms, affinity and specificity) of the antibody produced by 1D6 hybridoma cells were not altered by clinorotation. It is concluded that simulated microgravity affected the proliferating and adhesive, but not biosynthetic properties of hybridoma cells.

The beta-subunit composition of nicotinic acetylcholine receptors in the neurons of the guinea pig inferior mesenteric ganglion.

The antibodies against synthetic (183-192) fragments of beta2- and beta4-subunits of rat neuronal nicotinic acetylcholine receptor were used to study a beta-subunit composition of nicotinic receptors in the inferior mesenteric ganglion of the guinea pig by both immunocytochemical staining and blocking of excitatory postsynaptic potentials induced by electric stimulation of the pre-ganglionic nerve (intermesenteric trunk). The beta4-specific antibody stained 59.8 +/- 7.5% of neurons and inhibited the synaptic responses in all (n = 10) neurons studied by 25.5 +/- 1.8%. The beta2-specific antibody did not stain ganglionic neurons and did not affect the synaptic transmission. Taking into account the previously obtained data on the alpha-subunits found in this ganglion, it is concluded that the neurons of inferior mesenteric ganglion contain nicotinic receptors of alpha3(alpha5)beta4 subtypes involved in synaptic transmission through the intermesenteric tract.

Distribution of neuronal nicotinic acetylcholine receptors containing different alpha-subunits in the submucosal plexus of the guinea-pig.

The subunit composition and localisation of nicotinic acetylcholine receptors (nAChRs) in the submucosal plexus of the guinea-pig ileum were studied using both affinity-purified monoclonal and polyclonal antibodies against alpha3, alpha4, alpha5 and alpha7 nAChR subunits and specific alpha7-containing nAChRs blocker methyllycaconitine (MLA). By means of immunohistochemistry performed in non-dissociated preparations, it was found that only 4% of submucosal ganglia expressed nAChRs. Specific staining, associated with cell membranes, was found with alpha3-, alpha5- and alpha7-, but not alpha4-specific antibodies. Double staining using alpha5- and alpha7-specific antibodies demonstrated that about one-half of the nAChR-positive ganglia contained neurons immunoreactive to both antibodies, while the others possessed either alpha5- or alpha7-immunoreactivity. Nanomolar concentrations of MLA prevented alpha7-specific antibody binding and did not influence the alpha5-specific antibody binding even when applied in micromolar concentrations. In electrophysiological experiments performed using a patch-clamp 'whole-cell' recording method, the neurons were identified by their sensitivity to MLA. In conclusion, submucosal neurons of the guinea-pig ileum express nAChRs containing alpha3-, alpha5- and alpha7-subunits. The co localisation of alpha5- and alpha7-subunits found in immunohistochemical experiments as well as kinetic characteristics of MLA-blocked receptors found by electrophysiological experiments allow us to suggest the presence of heteromeric alpha7-containing nAChRs in the submucosal plexus of the guinea-pig ileum.

Functional nicotinic acetylcholine receptors are expressed in B lymphocyte-derived cell lines.

Nicotine has been shown to affect B lymphocyte immune response. In this study, we have explored the presence of nicotinic receptors in B lymphocyte-derived cell lines, myeloma X63-Ag8 and hybridoma 1D6. We found that myeloma expressed on average 10,170 +/- 1,100 [3H]epibatidine and 6,730 +/- 370 125I-alpha-bungarotoxin binding sites per cell, thus reflecting the presence of both homomeric and heteromeric nicotinic receptors. More specifically, the presence of alpha4- and alpha7-containing nicotinic receptor subunits was demonstrated in both myeloma and hybridoma cells with subunit-specific antibodies. It was significantly higher in dividing than in resting cells. Long-term exposure to nicotine, at physiological concentration found in smokers, resulted in up-regulation of both alpha4 and alpha7 subunits in hybridoma cells. Additionally, nicotine stimulated hybridoma cell proliferation, whereas it decreased antibody production. In contrast, alpha7-specific snake toxins inhibited cell proliferation but increased antibody production. It is concluded that myeloma and hybridoma cells express alpha4- and alpha7-containing nicotinic receptors, which participate in regulating cell proliferation and function.